Oligonucleotide protecting groups

ABSTRACT

Compounds of the invention having general formula (I)  
                 
 
     are useful reagents for protecting amine, guanidine, amidine or hydroxyl groups during organic synthesis. In particular, compounds are useful during oligonucleotide synthesis to protect nucleobase amines as well as tethered amines used for attaching functional moieties to oligonucleotides.

FIELD OF THE INVENTION

[0001] The present invention relates to protecting groups, moreparticularly to compounds useful for protecting amino, amidino,guanidino and hydroxyl groups, as well as methods of use thereof duringoligonucleotide synthesis.

BACKGROUND OF THE INVENTION

[0002] During oligonucleotide synthesis, convenient amino groupprotection methodology is important not only for exocyclic amines butalso for side chain amino groups (“aminolinkers” or “aminotethers”).These amino group-containing tethers can be conveniently deprotected andused to attach various functionalities to modify the biological orchemical properties of oligonucleotides (e.g., to conjugate groups whichcan improve uptake of antisense oligonucleotides by living cells); toattach chemical nucleases targeting the pathogenic genes; and to attachreporter groups (such as fluorescein or biotin) which are extensivelyused in DNA based diagnostics in following cellular trafficking ofantisense oligonucleotides (Manoharan, M., in Antisense Research andApplications, S. T. Crooke and B. Lebleu (eds.), CRC Press, Boca Raton,Fla., 1993, 303-349). In spite of their widespread use, the conventionalprotecting groups used in oligonucleotide chemistry for aminotethers areeither too labile during the monomer synthesis (e.g., CF₃CO—, Fmoc) orsomewhat inert, thereby requiring harsh conditions during, for example,oligonucleotide deprotection. The phthalimido group, for example,requires CH₃NH₂ in addition to the standard ammonium hydroxideconditions. The acid labile MMT (monomethoxytrityl) group is sometimesused, but generally to protect an aminolinker only at the 5′-end of theoligonucleotide.

[0003] To overcome these problems the alloc (allyloxycarbonyl) group(Kunz. H. Angew. Chem. 96, 426, 1984; Hayakawa, Y.; Wakabayashi, S.;Kato, H.; Noyori, R. J. Am. Chem. Soc. 112, 1691, 1990) has been adoptedas a protecting group for aminolinkers, as it can be removed usingzerovalent palladium (Pd (0)) either in solution phase or in solid phase(Barber-Peoch, I; Manoharan, M.; Cook, P. D. Nucleosides & Nucleotides16, 1407-1410, 1997; Nelson, P. S.; Muthini, S.; Kent, M. A; Smith T.H.; Nucleosides & Nucleotides 16, 1951-1959, 1997). The chloroformateCl—(C═O)—O—CH2—CH2—CN also has been used to protect nucleobase amines.Chloroformates, however, are unstable and difficult to use. It wouldtherefore be desirable to provide alternative reagents for protectingamine and other groups during synthesis.

SUMMARY OF THE INVENTION

[0004] In one aspect, the present invention provides compounds of theformula (I)

[0005] wherein

[0006] X is aryl or a covalent bond;

[0007] Y is aryl or a covalent bond;

[0008] R₁ is selected from succinimid-N-yl, phthalimid-N-yl,pyridin-N-yl, 4-nitophenyl, N-imidazol-1-yl, benzotriazol-2-yl,pyridin-2-yl, pentafluorophenyl, tetrafluorophenyl, triazol-N-yl,tetrazol-N-yl and norbornan-N-yl;

[0009] R₂ is cyano, nitro or CF₃;

[0010] R₃ and R₄ are each independently selected from the groupconsisting of H, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl andcycloalkyl-alkyl;

[0011] W is C(R₅) (R₆) or C(R₇)═C(R₇) where each R₅, R₆, and R₇ isindependently selected from the group consisting of H, alkyl, alkenyl,alkynyl, aryl, aralkyl, cycloalkyl and cycloalkyl-alkyl or both R₇substituents together form an unsaturated aromatic ring; and

[0012] n is an integer from 0 to 7.

[0013] In another aspect, the invention provide methods for protectingamine, guanidine, amidine or hydroxyl groups comprising reacting a freeamine, guanidine, amidine or phosphate with a compound according to thegeneral formula (I).

DETAILED DESCRIPTION OF THE INVENTION

[0014] R₁ can be selected from succinimid-N-yl, phthalimid-N-yl,pyridin-N-yl, 4-nitophenyl, -imidazol-1-yl, benzotriazol-2-yl,pyridin-2-yl, pentafluorophenyl, tetrafluorophenyl, triazol-N-yl,tetrazol-N-yl, pyrazol-N-yl and norbornan-N-yl. More preferably, R₁ issuccinimid-N-yl, phthalimid-N-yl or pyridin-N-yl, and even morepreferably succinimid-N-yl or phthalimid-N-yl. Most preferably, R₁ issuccinimid-N-yl.

[0015] R₂ can be cyano, nitro or CF₃. In a preferred embodiment, R₂ iscyano while X and Y are both a covalent bond. In another preferredembodiment R₂ is nitro while one of X and Y is aryl while the other is acovalent bond. In another embodiment R2 is cyano while Y is aryl (e.g.,1,4-phenylene) and X is a covalent bond.

[0016] R₃, R₄, R₅, R₆, and R₇ each be independently selected from thegroup consisting of H, alkyl, alkenyl, alkynyl, aryl, aralkyl,cycloalkyl and cycloalkyl-alkyl.

[0017] As used herein, the term “alkyl” includes but is not limited tostraight chain, branch chain, and cyclic unsaturated hydrocarbon groupshaving 1 to about 10 (preferably 1 to about 4) carbon atoms. “Alkenyl”includes but is not limited to straight chain, branch chain, and cyclicsaturated hydrocarbon groups having 2 to about 10 carbon atoms.“Alkynyl” includes but is not limited to hydrocarbon groups having 2 toabout 10 carbon atoms and a carbon-carbon triple bond. By “cycloalkyl”is meant mono- or bicyclic rings of 3 to 10 members optionallyunsaturated and optionaly attached to the carbon atom from which R₃ toR₇ depend by an alkyl chain thereby forming a “cycloalkyl” group. Whenany of R₃ through R₇ is alkenyl or alkynyl, the unsaturated bond orbonds preferably are spatially removed from the carbon atom from theydepend. In other words, the unsaturated bond is preferably not adjacentto said carbon atom.

[0018] “Aryl” is used herein interchangeably with “aromatic”, andincludes optionally substituted mono-, bi- and tricyclic, 5 to 14membered rings incorporating carbon atoms exclusively or incorporatingone or more heteroatoms such as N, O and S (as well as SO and SO₂),thereby forming a heteroaryl group. Prefered aryl groups include phenyl,naphthyl, pyridyl, quinolinyl, isoquinolinyl and naphthyridyl.

[0019] In preferred embodiments, R₃ through R₇ are independently H,alkyl or aryl. In a particularly prefered embodiment R₃, R₄ and R₅ are Hand R₆ is phenyl (preferably while n is 1). In another particularlyprefered embodiment, both R₃ and R₄ are H and both R₅ and R₆ are methyl(preferably while n is 1). In another preferred embodiment, each of R₃through R₆ are H (preferably while n is 1).

[0020] In other embodiments in which W includes olefinic moieties, R₂preferably is cyano or nitro. When W is olefinic, adjacent R₇substituents together may form an aromatic ring. Prefered aromatic(i.e., aryl) rings formed in this respect include benzene, naphthalene,pyridine and more preferably benzene.

[0021] X and Y preferably are both covalent bonds. In an alternateembodiment, one of X and Y is aryl (e.g., 1,4-phenylene) wherein X andCR₅R₆ are para to one another.

[0022] Compounds of the invention can be prepared according establishedorganic synthetic techniques. In a particular general method, compoundsare prepared by reacting an alcohol of formula (II) under suitableconditions (e.g. in acetonitrile/dichloromethane in the presence ofpyridine) with a dicarbonate of formula (III), wherein X, Y, n and R₁through R₇ are as previously defined. See Scheme 1 below. The alcohol(II) and dicarbonate (III) are either commercially available orthemselves are prepared from commercially available reagents accordingto established synthetic techniques.

[0023] Alternatively, compounds of formula (I) can be prepared byreacting a chloroformate of formula (IV) under suitable conditions withan alcohol of formula (V). See Scheme 2 below.

[0024] Again, chloroformate (IV) and alcohol (V) are either commerciallyavailable or themselves may be prepared from commercially availablereagents using established organic synthetic techniques.

[0025] The invention also provides methods for protecting amine,guanidine, amidine or hydroxyl groups comprising reacting a freereactive amine, guanidine, amidine, or hydroxyl with a compoundaccording to the general formula (I). Said amine, guanidine, amidine orhydroxyl group may be incorporated on or within various chemicalmolecular entities requiring protection, including but not limited toamino acids, peptides, proteins, nucleosides (RNA or DNA), nucleotidesand oligonucleotides. In particular, amino acids incorporating aminesand guanidines in their side chains (e.g., lysine and arginine,respectively) are suitable for methods of the invention. Other compoundsparticularly amenable to the present invention are nucleosides,nucleotides and oligonucleotides. For instance, exocyclic amine groupson nucleobases (e.g., cytosine, adenine and guanine) can be protectedaccording to methods of the invention. A class of nucleosides known as“peptide nucleic acids” (PNAs) incorporate a peptidic backbone in placeof a natural sugar-phosphate backbone, the amine group of which may alsobe protected according the method of the invention. Further, hydroxylgroups on the sugar of a nucleoside (e.g., at the 2′-, 3′- and 5′-positions) also can be protected.

[0026] In a particular embodiment, the amine, amidine, guanidine andhydroxyl groups that are protected according to the present inventionare located on tethering or linker groups. The amine, amidine, guanidineor hydroxyl groups on these tethers can, upon deprotection, be used toattach functional groups to nucleosides, nucleotides, and, inparticular, oligonucleotides to modify the biological or chemicalproperties of such moieties. Functional groups include conjugate groups(such as cholesterol) for enhancing cellular uptake, intercolators forenhancing hybridization, chemical nucleases for targeting pathogenicgenes, and reporter groups (such as fluorescein and biotin) fordiagnostic purposes or monitoring cellular trafficking. Tethers can beattached to the nucleobase, backbone or sugar moieties. In a particularembodiment, tethers are attached at the 2′-O position of a nucleosidesugar. Alternatively, tethers may be attached at the 3′-O position, forexample when oligonucleotides have one or more 2′-5′ linkage or at the5′-O position when the tether is at the 5′ terminus of anoligonucleotide.

[0027] Protecting groups may be subsequently removed to give the freeamine, amidine, quanidine or hydroxy by reacting with a suitablereagent. For amines, amidines and guanidines, suitable removing agentsinclude ammonium hydroxide (NH₄OH), triethylamine (NEt₃), DBU, Hunig'sbase, (iPr)2NEt, Et2N4, piperidine, morpholine, piperazine andpyrrolidine. For hydroxyl groups, suitable reagents for removingprotecting groups include DBU. Advantageously, one can achievedeprotection of all amine groups (nucleobase and tethered amines) andall phosphates of an oligonucleotide synthesis in a single step. This isaccomplished when a suitable phosphate protecting group is employed suchas β-cyanoethyl and a suitable deprotection reagent is used such asammonium hydroxide. Alternatively, the protecting group may be employedat specified groups and not at others, thereby affording selectiveprotection/deprotection ability.

[0028] In one aspect of the invention, compounds of formula (I) are usedin a guanylating process. A thiopseudourea of formula (VI) (wherein R′is a suitable protecting group such as alkyl, e.g. methyl) is reactedwith a dicarbonate of formula (III) to give a mixture of mono and bisprotected thiopseudourea (VII) and (VIII) (wherein Q is—X—(CR₅R₆)_(n)—CR₃R₄-Y-R₂). The bis compound (VIII) is then reacted witha primary amine RNH₂ wherein R is H or a guanidino protecting group suchas alkyl (e.g., methyl) or BOC to give a bis protected guanidino group(IX). Guanidino (IX) may then be used to add a guanidino functionalityto other compounds, in particular those with a reactive hydroxyl groupto give a protected functional group—NH—C(NH)—NH—CO—O—(CR₅R₆)N—CR₃R₄-Y-R₂. Deprotection of the guanidinylgroup may achieved using a suitable reagent including base.

[0029] Additional objects, advantages, and novel features of thisinvention will become apparent to those skilled in the art uponexamination of the following examples thereof, which are not intended tobe limiting.

EXAMPLE 1

[0030] Preparation of cyanoethyloxycarbonyloxy succinimide (compound 1)

[0031] To 3.0 g of 2-cyanoethanol (42.21 mmol) in 140 mL anhydrousCH₃CN, 17 g of N,N′-disuccinimidyl carbonate (Fluka, 66 mmol) was addedfollowed by 5.5 mL of pyridine. The suspension became a clear solutionafter about 1 hr. The solution was shaken for an additional 1 hr, andthen evaporated. The solution was redissolved in 100 mL of CH₂Cl₂,washed with 5% NaHCO₃ solution followed by saturated NaCl solution. Theorganic layer was dried over anhydrous MgSO₄ and evaporated to give 8.5g of a creamy white solid (95% crude yield). The crude product waspurified in silica gel using CH₂Cl₂:EtOAc (50:50) to give 7.2 g of awhite crystalline compound 1. (Rf=0.21) ¹H NMR (CDCl₃) (2.85, s, 4H;2.75 t, —CH₂—CN; 4.45, t, —CH₂—O—) M.P. 104.1° C.

[0032]¹³C NMR (DMSO); 169.82 (N—C≡O) 150.91 (—O—C(═O)—O) 117.91 (C═N)65.86 (—CH₂—O—) 25.39 (—CH—C—) 17.33 (—CH₂CN).

EXAMPLE 2

[0033] Preparation of 2′-O-aminoethyl-5′-O-DMT-5-methyluridine (compound4)

[0034] N-(2-hydroxyethyl)phthalimide (277 g, 1.45 mol) was slowly addedto a solution of borane in tetrahydrofuran (1M, 600 mL) with stirring.Hydrogen gas evolved as the solid dissolved. Once the rate of bubblingsubsided, the solution was placed in a 2L stainless steel bomb.2,2′-anhydro-5-methyluridine (60 g, 0.25 mol) and sodium bicarbonate(120 mg) were added and the bomb was sealed. After 30 minutes, the bombwas vented for the last time and then placed in an oil bath and heatedto 150° C. internal temperature for 24 h. The bomb was then cooled toroom temperature and opened. TLC revealed all the starting material wasgone. The crude solution was concentrated and the residue was colummnedon silica gel starting with straight ethyl acetate to remove excessphthalimide reagent followed by ethyl acetate-methanol 95/5 to elute theproduct to give 22.2 g (20.6%) of ca 90% pure product 2.

[0035] 2′-O-phthalimidoethyl-5-methyluridine 2 (22.2 g, 0.053 mol) wascoevaporated with pyridine (2×75 mL) and then dissolved in 100 mL ofpyridine. Dimethoxytrityl chloride (27 g, 0.080 mol) was added in oneportion with stirring. TLC after 1 h indicated a complete reaction.Methanol (10 mL) was added to quench the reaction. The reaction wasconcentrated and the residue partitioned between ethyl acetate andsaturated sodium bicarbonate solution (150 mL each). The organic layerwas concentrated and the residue was dissolved in a minimum amount ofdichloromethane and applied on a silica gel column. The compound waseluted with ethyl acetate-hexanes-triethylamine (50:50:1 to 80:20:1) togive 26.1 g (82%) of pure product2′-O-phthalimidoethyl-5′-O-DMT-5-methyluridine 3.

[0036] 2′-O-phthalimidoethyl-5′-O-DMT-5-methyluridine 3 (21.1 g, 0.29mol) was dissolved in methanol (500 mL). Anhydrous hydrazine (4.9 mL,0.15 mol) was added and the solution was heated to reflux. TLC after 3 hindicated a complete reaction. The solution was concentrated andcolumned on silica gel using methanol and then methanol-ammoniumhydroxide (98:2) to give 10.4 g of pure product2′-O-aminoethyl-5′-O-DMT-5-methyluridine 4 as a white foam and 2 g ofslightly contaminated product (total yield 12.4 g, 71%).

EXAMPLE 3

[0037] Preparation of2′-O-[N-cyanoethyloxy-carbonyl-2-(aminoethyl)]-5′-O-dimethoxy-trityl-5-methyluridine(compound 5)

[0038] To cyanoethyloxycarbonyloxy-N-succinimide 1 in 10 mL of CH₂Cl₂was added 0.5 mL of pyridine followed by 1.43 g of2′-O-(aminoethyl)-5′-O-DMT-5-methyluridine (compound 4, 2.35 mmol) andstirred for 1 hr. TLC (CH₂Cl₂/CH₃OH 9:1; Rf=0.48) indicated completeconversion of amine to the carbamate derivative. The mixture was thendiluted with CH₂Cl₂ (50 mL) and washed successively with aqueous NaHCO₃solution, saturated NaCl solution and dried over MgSO₄. Chromatographyover silica and elution with CH₂Cl₂:EtoAc gave the product 5 (1.2 g,73%).

EXAMPLE 4

[0039] Preparation of2′-O-[N-cyanoethyloxy-carbonyl-2-(aminoethyl)]-3′-O-[N,N-diisopropyl-aminocyano-ethyloxyphosphoramidite]-5′-O-DMT-5-methyluridine(compound 6)

[0040] 5′-O-DMT-2′-O-(N-cyanoethyloxy carbonylaminoethyl)-5-methyl-uridine 5 (700 mg, 1 mmol) was dissolved in 15 mLof dry CH₂Cl₂ and to this solution 85 mg of diisopropylaminotetrazoliumsalt (0.5 mmol) followed by 420L of 2-cyanoethyl-N,N,N′N′-tetraisopropylphosphoramidite was added slowly using a syringe under argon. Themixture was stirred at room temperature over night and in the morningthe TLC indicated almost complete. 40 μL of the phosphitylation reagentwas added and stirred for an additional 2 hrs. TLC then indicatedcomplete conversion of the starting material to the phosphoramidite(CH₂Cl₂:EtOAc 50:50; Rf=0.33). The reaction mixture was diluted with 50mL CH₂Cl₂, washed with saturated NaHCO₃ solution followed by saturatedNaCl solution. The organic layer was dried over MgSO₄ and evaporated todryness. The crude foam was purified in silica gel and eluted with 50:50ethylacetate:CH₂Cl₂ to give compound 6. Yield=720 mg (81%) ³¹P NMR:CDCl₃ (149.5ppm and 150.5ppm)

EXAMPLE 5

[0041] Solid phase synthesis of oligonucleotides incorporating CEOCprotected 2′-O-aminoethyl nucleosides

[0042]2′-O-[N-cyanoethyloxycarbonyl-2-(aminoethyl)]-3′-O-[N,N-diisopropyl-aminocyano-ethyloxyphosphoramidite]5′-O-DMT-5-methyl uridine (compound 6, 192 mg, 0.2 mmol)was dissolved in 2 mL of anhydrous acetonitrile and loaded onto anExpedite Nucleic Acid Synthesis system (Millipore) to synthesizeoligonucleotides incorporating CEOC-amino protected nucleosides. Theamidite concentration was 0.1M. The coupling efficiencies were more than90%. When coupling the first amidite, the coupling time was extended to10 min. and the step carried out twice. All other steps in the protocolsupplied by Millipore were used. Oligonucleotides were cleaved from thecontrolled pore glass (CPG) support and deprotected (including CEOCgroups) under standard conditions using concentrated aqueous NH₄OH (30%)at 55° C. 5′-O-DMT-containing oligonucleotides were then purified byreverse phase high performance liquid chromatography (C-4, Waters,7.8×300 mm, A-100 mm triethylammonium acetate, pH 7; B=acetonitrile;8-18% of B in 30 minutes; flow 1.5 mL/min.). Detritylation with aqueous80% acetic acid and evaporation, followed by desalting in a SephadexG-25 column gave oligonucleotides incorporating 2′-O-aminoethylnucleotides which were analyzed by CGE and mass spectrometry (see Table1).

EXAMPLE 6

[0043] Preparation of2′-O-[N-cyanoethyloxy-carbonyl-(6-aminohexyl)]-5′-O-dimethoxy-trityl-5-methyl-uridine(compound 7)

[0044] To 3.3 g of 2′-O-(6-aminohexyl)-5′-O-dimethoxytrityl-5-methyluridine (prepared according to the same procedure as for compound 4) (5mmol) in 20 mL of anhydrous CH₂Cl₂, 1 mL of pyridine was added followedby 1.2 g (5.6 mmol) of 2-cyanoethyloxycarbonyloxy succinimide. Thereaction mixture was stirred for 2 hrs and tested for completion ofreaction by TLC (CH₂Cl₂:CH₃OH 9:1). Reaction mixture was applied tosilica gel equilibrated with CH₂Cl₂:CH₃OH 9:1 and eluted with the same(Rf=0.51). Yield=3.13 g, 82%. ¹H and ¹³C NMR affirmed the expectedcompound 7.

EXAMPLE 7

[0045] Preparation of5′-O-DMT-2′-O-[N-cyanoethyloxy-carbonyl-(6-aminohexyl)]-5-methyluridine-3′-O-(N,N-diisopropylamino-2-cyanoethyloxy)phosphoramidite(compound 8)

[0046]2′-O-[N-cyanoethyloxycarbonyl-(6-aminohexyl)]-5′-O-dimethoxytrityl-5-methyl-uridine7 (1.51 g, 2 mmol) was dissolved in 30 mL of anhydrous CH₂Cl₂ and tothis solution 170 mg of diisopropylamino tetrazolium salt (1 mmole)followed by N,N,N′N′-tetraisopropyl phosphoramidite (990 μL, 2.6 mmols)under argon atmosphere. The reaction mixture was stirred for 16 hrs. TLCanalysis (50:50 CH₂Cl₂/ethylacetate) indicated completion of thereaction. The reaction mixture was then diluted with 100 mL of CH₂Cl₂,extracted with saturated NaHCO₃ solution, washed with saturated NaClsolution and dried over MgSO₄. Evaporation to dryness gave a white foamwhich was applied on the top of silica gel made with CH₂Cl₂ containing0.1% pyridine. The amidite was loaded in CH₂Cl₂ and eluted with 40:60CH₃COOEt/CH₂Cl₂ to give 1.3 g of purified amidite 8 (68% yield). ³¹PNMR=150.5, 151 ppm.

EXAMPLE 8

[0047] Solid phase synthesis of oligonucleotides incorporating CEOCprotected 2′-O-aminohexyl nucleosides

[0048] 5′-O-DMT-2′-O-[N-cyanoethyloxy-carbonyl-(6-aminohexyl)]-5-methyluridine-3′-O-(N,N-diisopropylamino-2-cyanoethyloxy) phosphoramidite(compound 8) (400 mg, 0.39 mmol) was dissolved in 3.9 mL of anhydrousacetonitrile and loaded onto an Expedite Nucleic Acid Synthesis system(8909) (Millipore) to synthesize the oligonucleotides. The amiditeconcentration was 0.1M. The coupling efficiencies were more than 90%.When coupling the first amidite, the coupling time was extended to 10min. and this step was carried out twice. All other steps in theprotocol supplied by Millipore were used. Oligonucleotides oligomerswere cleaved from the controlled pore glass (CPG) supports anddeprotected (including cyanoethyloxycarbonyl protecting groups) understandard conditions using concentrated aqueous NH₄OH (30%) at 55° C.5′-O-DMT-containing oligomers were then purified by reverse phase highperformance liquid chromatography (C-4, Waters, 7.8×300 mm, A=50 mMtriethylammonium acetate, pH −7, B=acetonitrile, 5-60% of B in 60 min.,flow 1.5 mL/min.). Detritylation with aqueous 80% acetic acid andevaporation, followed by desalting in a Sephadex G-25 column gaveoligonucleotides incorporating 2′-O-aminohexyl nucleotides (see Table 1)which were analyzed by HPLC, CGE and mass spectrometry.

EXAMPLE 9

[0049] 6-(N-2-cyanoethyloxycarbonyl)aminohexanol (compound 9)

[0050] 6-Aminohexanol (0.5 g, 4.23 mmol) was dissolved in anhydrousCH₂Cl₂ (10 mL). Compound 1 (1.08 g, 5.09 mmol) was added and stirred for2 hrs. The reaction was followed by TLC (5% MeOH in CH2Cl2). Solvent wasremoved under vacuum and residue was placed on a flash column and elutedwith 5% MeOH in CH₂Cl₂ to get 9 as a white powder (0.883 g, 96% yield).Rf (0.28, 5% MeOH in CH₂Cl₂). ¹H NMR (CDCl₃) δ4.9 (br, 1H), 4.3 (t, 2H,J=6.12 Hz), 3.68 (3H, t, J=3.71 Hz), 2.73 (2H, t, J=6.12 Hz), 1.6-1.39(m, 8H). ¹³C NMR (CDCl₃) d 155.64 (C═O), 117.34 (C≡N), 62.49, 59.01,40.92, 32.47, 29.73, 26.35, 25.30, 18.49. MS (APCI⁺) calculated forC₁₀H₁₉O₃N₂ ⁺215; observed 215.1.

EXAMPLE 10

[0051]6-(N-2-cyanoethyloxycarbonyl)aminohexyl-β-cyanoethyl-N,N-diisopropylphosphoramidite 10

[0052] Compound 9 (0.72 g, 3.36 mmol) was mixed with diisopropylaminetetrazolide (0.29 g, 1.68 mmol). The mixture was then dried over P₂O₅under high vacuum overnight at 40° C. The reaction mixture was flushedwith argon. Anhydrous acetonitrile (16.95 mL) was added, followed bydropwise addition of 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.52 g, 5.04 mmol). The reaction mixture was stirredat room temperature for 4 hrs under argon atmosphere. Solvent wasremoved under vacuum. The residue was placed on a flash column andeluted with ethylacetate:hexane 1:1 to get compound 10 as an oil (44%yield). Rf (0.05, ethylacetate:hexane, 1:1) ¹H NMR (CDCl₃) δ4.29 (t, 2H,J=6.24 Hz), 3.53-3.95 (m, 6H), 3.21 (Q, 2H, J=6.46, 6.78), 2.62-2.75 (m,4H), 1.48-1.7 (M, 8H), 1.22 (S, 6H), 1.22 (S, 6H). ³¹P NMR (CDCl₃)δ147.78, MS (FAB⁺) calculated for C₁₉O₄H₃₅NAPNa⁺=437; observed 437.

EXAMPLE 11

[0053] Solid phase synthesis of oligonucleotides incorporating CEOCprotected aminohexanol

[0054] Amidite 10 (0.060 g, 0.14 mmol) was dissolved in anhydrousacetonitrile (1.4 mL) and loaded onto an Expedite Nucleic Acid SynthesisSystem (Millipore) to synthesize the oligonucleotide. Commerciallyavailable dA, dC, T and dG amidites were used to synthesizeoligonucleotide. Coupling efficiencies were more than 95%. All othersteps in the protocol for 1 mmol DNA synthesis supplied by Milliporewere used except the extended coupling time (10 min.) for amidite 10.The oligomers were cleaved from the controlled pore glass (CPG) supportsand deprotected under standard conditions using concentrated aqueousNH₄OH (30%) at 55° C. Fully deprotected oligonucleotide was thenpurified by HPLC on reverse phase column (C-4, Waters, 7.8×300 mm, A-50mm triethylammonium acetate, pH 7; B=acetonitrile, 5-60% B in 60 mins.,flow 2.5 mL/min.). Purity and integrity of oligonucleotide(oligonucleotide 7 in table I) was established by CGE, HPLC and massspectrometry. Electrospray mass calculated for M⁺=6161.68; observedM⁺=6160.72.

EXAMPLE 12

[0055] Conjugation to fluorescein

[0056] Oligonucleotides 4 and 7 (see table 1 below) containing thetethered amino functionality were used to conjugate fluorescein to theoligonucleotide. Purified oligonucleotides (15OD) were taken in 1MNaHCO₃/Na₂CO₃ buffer (100 μL, pH9.2). A solution of fluoresceinisothiocyanate (100 μL, 1M solution in DMF) was added to the solution ofoligonucleotides and kept at room temperature for 24 hrs. Unreactedfluorescein isothiocyanate was removed by passing the reaction mixturethrough a Sephadex G-25 and eluting the column with water. Conjugatedoligonucleotides were then purified by reverse phase high performanceliquid chromatography and characterized by mass spectrometry andanalytical HPLC.

EXAMPLE 13

[0057] Guanidium protection

[0058] 2-Methyl-2-thiopseudourea.1/2H₂SO₄ (280 mg, 2 mmols) wassuspended in 15 mL of CH₂Cl₂ and 15 mL of 10% NaHCO₃.Cyanoethyloxycarbonyloxy succinimide 1 (900 mg, 4.2 mmols) was added andafter stirring overnight at room temperature for 2 hrs. The organiclayer was separated. The water layer was extracted (3×50 ml) with CH₂Cl₂and the combined organic layers were dried over MgSO₄. TLC indicated twoproducts. The dried organic solution was evaporated and eluted with 5%ethyl acetate in methylene chloride followed by 10% ethyl acetate inmethylene chloride. The fast moving compound was the desired bis CEOCcompound (N,N′-bis-CEOC-2-methyl-2-thiopseudourea) and the slow movingcompound is the mono CEOC compound. Yield of 6=520 mg.

[0059] Primary amine was dissolved in DMF (1 mmol in 3 mL).Triethylamine (1 eq.) and N,N′-bis CEOC-2-methyl-2-thiopseudourea (1.1equivalent) were added. After stirring for 3 hrs at room temp, TLCindicated completion of reaction and water was added. The mixture wasextracted with ethyl acetate and purified by silica gel columnchromatography.

EXAMPLE 14

[0060] Preparation of cyanoethyloxycarbonyloxy pyridine-2-yl

[0061] To a solution of β-cyanoethanol (0.3 g, 4.22 mmol) in CH₂Cl₂(31.7 mL), 13, di(2-pyridyl)carbonate (1.37 g, 6.34 mmol) was added(Ghosh, A. K.; Duong, T. McKee, S. P. Tetrahedron Lett. 32, 4251, 1991).Triethylamine (0.8 mL, 6.34 mmol) was added to the above solution andthe reaction mixture was stirred at ambient temperature for 8 hrs. TLCshowed disappearance of starting material. Solvent was removed undervacuum to give residue 11. TABLE 1 HPLC Oligo Oligo Mass ret. no.sequence 2′-O mod. exp. obs. time^(c) 1 GAT*CT^(d) aminohexyl 1895.001895.57^(a) 20.05 2 T*CCAGGT*GT* CCGCAT*C aminohexyl 5599.00 5597.24^(a)24.01 3 CTCGTACT*T*T *T*CCGGTCC aminohexyl 5853.21 5854.56^(b) 20.25 4CTAGTACCT*TT CCGGTCC aminohexyl 5493.21 5493.91^(b) 16.47 5 GAT*CTaminoethyl 1837.00 1837.00^(a) 19.92 6 T*CCAGGT*GT* aminoethyl 5368.005370.40 23.88 CCGGCAT*C 7 LTGCATCCCCCA GGCCACCAT^(f) — 6161.68 6160.7217.46^(e) 8 CTCGTACCT*TT  aminohexyl- CCGGTCC CS—NH— 5881.60 5880.8922.08^(e) fluoroscein 9 L_(FL)TGCATCCCC — 6550.68 6550.01 23.74^(e)CAGGCCACCAT^(g)

[0062] Those skilled in the art will appreciate that numerous changesand modifications may be made to the preferred embodiments of theinvention and that such changes and modifications may be made withoutdeparting from the spirit of the invention. It is therefore intendedthat the appended claims cover all such equivalent variations as fallwithin the true spirit and scope of the invention.

We claim:
 1. A compound of general formula (I):

wherein X is aryl or a covalent bond; Y is aryl or a covalent bond; R₁is selected from succinimid-N-yl, phthalimid-N-yl, pyridin-N-yl,4-nitophenyl, N-imidazol-1-yl, benzotriazol-2-yl, pyridin-2-yl,pentafluorophenyl, tetrafluorophenyl, triazol-N-yl, tetrazol-N-yl andnorbornan-N-yl; R₂ is cyano, nitro or CF₃; R₃ and R₄ are eachindependently selected from the group consisting of H, alkyl, alkenyl,alkynyl, aryl, aralkyl, cycloalkyl and cycloalkyl-alkyl; W is C(R₅) (R₆)or C(R₇)═C(R₇) where each R₅, R₆, and R₇ is independently selected fromthe group consisting of H, alkyl, alkenyl, alkynyl, aryl, aralkyl,cycloalkyl and cycloalkyl-alkyl or both R₇ substituents together form anunsaturated aromatic ring; and n is an integer from 0 to
 7. 2. Acompound according to claim 1 , wherein Y is a covalent bond.
 3. Acompound according to claim 2 , wherein X is a covalent bond.
 4. Acompound according to claim 3 , wherein R₃ and R₄ are both H.
 5. Acompound according to claim 1 , wherein R₅ and R₆ are independentlyselected from H, alkyl and aryl.
 6. A compound according to claim 5 ,wherein R₅ and R₆ are independently selected from H, methyl and phenyl.7. A compound according to claim 6 , wherein R₅ and R₆ are both methyl.8. A compound according to claim 6 , wherein R₅ is H and R₆ is phenyl.9. A compound according to claim 6 , wherein R₅ and R₆ are both H.
 10. Acompound according to claim 1 , which is cyanoethyloxycarbonyloxysuccinimide.
 11. A compound according to claim 1 , wherein Y is aryl.12. A compound according to claim 11 , wherein R₂ is nitro.
 13. Acompound according to claim 11 , wherein Y is 1,4-phenylene.
 14. Acompound according to claim 11 , wherein X is a covalent bond.
 15. Acompound according to claim 14 , wherein R₃, R₄, R₅ and R₆ are each H.16. A compound according to claim 15 , wherein R, is succinimide orpyridine-2-yl.
 17. A compound according to claim 16 , wherein R, issuccinimide.
 18. A method of protecting amine, guanidine, amidine orhydroxyl groups comprising reacting a free amine, guanidine or amidinewith a compound according to claim 1 .
 19. The method according to claim18 , wherein said amine, guanidine, amidine or hydroxyl group is asubstituent of a nucleic acid.
 20. The method according to claim 19 ,wherein said nucleic acid is a nucleoside.
 21. The method according toclaim 19 , wherein said nucleic acid is a nucleotide.
 22. The methodaccording to claim 19 , wherein said nucleic acid is an oligonucleotide.23. The method according to claim 19 , wherein said amine is asubstituent of a base of said nucleic acid.
 24. The method according toclaim 19 , wherein said amine, guanidine, amidine or hydroxyl is asubstituent on a sugar of said nucleic acid.
 25. The method according toclaim 19 , wherein said amine, guanidine, amidine or hydroxyl is asubstituent on a tethering group attached to a 2′ position of a sugar ofsaid nucleic acid.
 26. A method of protecting amine groups comprisingreacting a free amine with a compound according to claim 1 .
 27. Amethod of protecting guanidine groups comprising reacting a freeguanidine with a compound according to claim 1 .
 28. A method ofpreparing a compound of formula (IX)

wherein R is H, or a guanidino protecting group; Q is—X—(CR₅R₆)_(n)—CR₃R₄-Y-R₂; X is aryl or a covalent bond; Y is aryl or acovalent bond; R₁ is selected from succinimid-N-yl, phthalimid-N-yl,pyridin-N-yl, 4-nitophenyl, -imidazol-1-yl, benzotriazol-2-yl,pyridin-2-yl, pentafluorophenyl, tetrafluorophenyl, triazol-N-yl,tetrazol-N-yl and norbornan-N-yl; R₂ is cyano, nitro or CF₃; R₃ and R₄are each independently selected from the group consisting of H, alkyl,alkenyl, alkynyl, aryl, aralkyl, cycloalkyl and cycloalkyl-alkyl; W is C(R₅) (R₆) or C(R₇)═C(R₇) where each R₅, R₆, and R₇ is independentlyselected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl,aralkyl, cycloalkyl and cycloalkyl-alkyl or both R₇ substituentstogether form an unsaturated aromatic ring; and n is an integer from 0to 7; comprising the steps of: reacting a compound of formula (VIII)

with an amine, wherein R′ is a thiopseodourea protecting group.
 29. Acompound of formula (IX) or formula (VIII):

wherein R is H, or a guanidino protecting group; Q is—X—(CR₅R₆)—CR₃R₄-Y-R₂; X is aryl or a covalent bond; Y is aryl or acovalent bond; R₁ is selected from succinimid-N-yl, phthalimid-N-yl,pyridin-N-yl, 4-nitophenyl, N-imidazol-1-yl, benzotriazol-2-yl,pyridin-2-yl, pentafluorophenyl, tetrafluorophenyl, triazol-N-yl,tetrazol-N-yl and norbornan-N-yl; R₂ is cyano, nitro or CF₃; R₃ and R₄are each independently selected from the group consisting of H, alkyl,alkenyl, alkynyl, aryl, aralkyl, cycloalkyl and cycloalkyl-alkyl; W isC(R₅) (R₆) or C(R₇)═C(R₇) where each R₅, R₆, and R₇ is independentlyselected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl,aralkyl, cycloalkyl and cycloalkyl-alkyl or both R₇ substituentstogether form an unsaturated aromatic ring; n is an integer from 0 to 7;and R′ is a thiopseodourea protecting group.